Simultaneous Analysis of Tramadol, O-Desmethyltramadol, and N-Desmethyltramadol Enantiomers in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)-trans-T and (-)-trans-T. The main metabolic pathways are O-demethylation and N-demethylation, producing trans-O-desmethyltramadol (M1) and trans-N-desmethyltramadol (M2) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)-trans-T and (+)-M1 and to the monoaminergic action of (+/-)-trans-T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans-T, M1, and M2 enantiomers. The analytes were resolved on a Chiralpak (R) AD column using hexane: ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans-T and M1 and 0.1 ng/ml for M2. The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans-T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans-T and M2 was enantioselective (AUC((+)/(-)) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans-T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans-T pharmacokinetics. Chirality 23: 287-293, 2011. (C) 2010 Wiley-Liss, Inc.

Evaluation of the genotoxicity of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes

In the present study, experiments were carried out to evaluate the mutagenic potential and genotoxic effects of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes, using the micronucleus and comet assays. Significant damage to DNA was observed for crotoxin and crotapotin (CA). Basic phospholipase A(2) (CB) and crotamine did not present any mutagenic potential when evaluated by the micronucleus test. C. d. terrificus crude venom was able to induce the formation of micronuclei, similarly to the mutagenic drug used as a positive control. In the comet assay, all the toxins tested (crotamine, crotoxin, CB and CA) and C. d. terrificus venom presented genotoxic activity. Studies on the cytogenetic toxicology of animal venoms and their isolated proteins are still very scarce in the literature, which emphasizes the importance of the present work for the identification and characterization of potential therapeutic agents, as well as for the better understanding of the mechanisms of action of toxins on the human body. (C) 2011 Elsevier B.V. All rights reserved.

The degree of external genitalia virilization in girls with 21-hydroxylase deficiency appears to be influenced by the CAG repeats in the androgen receptor gene

Background Women with 21-hydroxylase deficiency present much variability in external genitalia virilization, even among those with similar impairments of 21-hydroxylase (21OH) activity. Objective To evaluate if the number of CAG (nCAG) repeats of the androgen receptor gene influences the degree of external genitalia virilization in women with CYP21A2 mutations, grouped according to impairment of 21OH activity. Patients The nCAG was determined in 106 congenital adrenal hyperplasia (CAH) patients and in 302 controls. The patients were divided, according to their CYP21A2 genotypes, into Groups A and B, which confer total and severe impairment of 21OH activity, respectively. Methods The inactivation pattern of the X-chromosome was studied through genomic DNA digestion with Hpa II. The CAG repeat region was amplified by polymerase chain reaction (PCR) and analysed by GeneScan. Results The nCAG and the frequency of severe skewed X-inactivation did not differ between normal women and patients. The nCAG median in genotype A was 20.7 (IQR 2.3) for Prader I + II, 22.5 (3.6) for Prader III and 21 (2.9) for Prader IV + V (P < 0.05 for Prader III and Prader IV + V). The nCAG median in genotype B was 21.3 (1.1) for Prader I + II, 20.5 (2.9) for Prader III and 22 (2.8) for Prader IV + V (P > 0.05). A significant difference was found regarding the nCAG median in patients presenting Prader III from genotypes A and B. Conclusions We observed great variability in the degree of external genitalia virilization in both CYP21A2 genotypes, and we showed that the CAG repeats of the androgen receptor gene influences this phenotypic variability.

Is the ultrasound-estimated bladder weight a reliable method for evaluating bladder outlet obstruction?

OBJECTIVE To evaluate the correlation between ultrasound-estimated bladder weight (UEBW) in patients with different degrees of bladder outlet obstruction (BOO). METHODS We evaluated 50 consecutive non-neurogenic male patients with lower urinary tract symptoms (LUTS) referred to urodynamic study (UDS). All patients self-answered the International Prostate Score Symptoms (IPSS) questionnaire. After the UDS, the bladder was filled with 150 mL to determine UEBW. Patients with a bladder capacity under 150 mL, a previous history of prostate surgery or pelvic irradiation, an IPSS score <8, a bladder stone or urinary tract infection were excluded. After a pressure-flow study, the Schafer linear passive urethral resistance relation nomogram was plotted to determine the grade of obstruction: Grades I-II/VI were defined as mild obstruction, Grades III-IV/VI as moderate obstruction, and Grades V-VI/VI as severe obstruction. RESULTS The UEBW was 51.7 +/- 26.9, 54.1 +/- 30.0 and 54.8 +/- 28.2 in patients with mild, moderate and severe BOO, respectively (P = 0.130). The UEBW allowed us to define four groups: (i) UEBW < 35 g; (ii) 35 g <= UEBW < 50 g; (iii) 50 g <= UEBW < 70 g; and (4) UEBW >= 70 g. We did not find any differences in age, prostate weight, IPSS, PVR, cystometric bladder capacity, presence of detrusor overactive and degree of obstruction in the aforementioned groups. CONCLUSION Despite the fact that some studies have emphasized the value of UEBW as an efficient non-invasive method for evaluating lower urinary tract obstruction, our study suggests that UEBW does not present any individual correlation with LUTS or objective measurements of BOO.

Low birth weight in response to salt restriction during pregnancy is not due to alterations in uterine-placental blood flow or the placental and peripheral renin-angiotensin system

A number of studies conducted in humans and in animals have observed that events occurring early in life are associated with the development of diseases in adulthood. Salt overload and restriction during pregnancy and lactation are responsible for functional (hemodynamic and hormonal) and structural alterations in adult offspring. Our group observed that lower birth weight and insulin resistance in adulthood is associated with salt restriction during pregnancy On the other hand, perinatal salt overload is associated with higher blood pressure and higher renal angiotensin II content in adult offspring. Therefore, we hypothesised that renin-angiotensin system (RAS) function is altered by changes in sodium intake during pregnancy. Such changes may influence fetoplacental blood flow and thereby fetal nutrient supply, with effects on growth in utero and, consequently, on birth weight. Female Wistar rats were fed low-salt (LS), normal-salt (NS), or high-salt (HS) diet, starting before conception and continuing until day 19 of pregnancy, Blood pressure, heart rate, fetuses and dams` body weight, placentae weight and litter size were measured on day 19 of pregnancy. Cardiac output, uterine and placental blood flow were also determined on day 19. Expressions of renin-angiotensin system components and of the TNF-alpha gene were evaluated in the placentae. Plasma renin activity (PRA) and plasma and tissue angiotensin-converting enzyme (ACE) activity, as well as plasma and placental levels of angiotensins I, II, and 1-7 were measured. Body weight and kidney mass were greater in HS than in NS and LS dams. Food intake did not differ among the maternal groups. Placental weight was lower in LS dams than in NS and HS dams. Fetal weight was lower in the US group than in the NS and HS groups. The PRA was greater in IS dams than in NS and HS dams, although ACE activity (serum, cardiac, renal, and placental) was unaffected by the level of sodium intake. Placental levels of angiotensins I and II were lower in the HS group than in the ISIS and IS groups. Placental angiotensin receptor type 1 (AT(1)) gene expression and levels of thiobarbituric acid reactive substances (TBARS) were higher in HS dams, as were uterine blood flow and cardiac output. The degree of salt intake did not influence plasma sodium, potassium or creatinine. Although fractional sodium excretion was higher in HS dams than in NS and LS dams, fractional potassium excretion was unchanged. In conclusion, findings from this study indicate that the reduction in fetal weight in response to salt restriction during pregnancy does not involve alterations in uterine-placental perfusion or the RAS. Moreover, no change in fetal weight is observed in response to salt overload during pregnancy. However, salt overload did lead to an increase in placental weight and uterine blood flow associated with alterations in maternal plasma and placental RAS. Therefore, these findings indicate that changes in salt intake during pregnancy lead to alterations in uterine-placental perfusion and fetal growth. (C) 2008 Elsevier Inc. All rights reserved.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

The study reported here is a classical bottom-up proteomic approach where proteins from wasp venom were extracted and separated by 2-DE; the individual protein spots were proteolytically digested and subsequently identified by using tandem mass spectrometry and database query with the protein search engine MASCOT. Eighty-four venom proteins belonging to 12 different molecular functions were identified. These proteins were classified into three groups; the first is constituted of typical venom proteins: antigens-5, hyaluronidases, phospholipases, heat shock proteins, metalloproteinases, metalloproteinase-desintegrin like proteins, serine proteinases, proteinase inhibitors, vascular endothelial growth factor-related protein, arginine kinases, Sol i-II and -II like proteins, alpha-glucosidase, and superoxide dismutases. The second contained proteins structurally related to the muscles that involves the venom reservoir. The third group, associated with the housekeeping of cells from venom glands, was composed of enzymes, membrane proteins of different types, and transcriptional factors. The composition of P. paulista venom permits us to hypothesize about a general envenoming mechanism based on five actions: (i) diffusion of venom through the tissues and to the blood, (ii) tissue, (iii) hemolysis, (iv) inflammation, and (v) allergy-played by antigen-5, PLA1, hyaluronidase, HSP 60, HSP 90, and arginine kinases.

Mandibular Function is Severely Impaired in Systemic Sclerosis Patients

Aims: To evaluate the presence of temporomandibular disorders (TMD) in systemic sclerosis (SSc) patients and its possible association with the severity of skin involvement. Methods: The presence of TMD was evaluated in 35 SSc women and 30 age- and sex-matched healthy controls by means of the anamnestic (A(i)) and clinical (D(i)) Helkimo indices; the jaw mobility was further analyzed (M(I)). Skin involvement was scored by the Modified Rodnan Skin Score (MRSS). Results: Signs and symptoms of TMD were more frequent in SSc patients than in controls, the frequency distribution of the different clinical dysfunction indices differing significantly (P < .001) between patients (D(i)0 8.6%, D(i)I 48.6%, D(i)II 22.8%, and D(i)III 20%) and controls (D(i)0 50%, D(i)I 33.3%, and D(i)II 16.7%). Cyclophosphamide for severe and rapidly progressive cutaneous fibrosis was prescribed in six out of seven patients with severe signs (D(i)III), in contrast this treatment was indicated for only two out of 25 patients with mild to moderate signs (D(i)I and D(i)II, P <. 001). Impaired jaw mobility was more frequent in SSc patients than controls (P < .001). It was severe in 77.1% (M(I)II) and mild in 22.9% (M(1)I) of the cases, in contrast to controls (M(I)0 33.4%, M(I)I 53.3%, and M(I)II 13.3%; P < .001). Approximately half of SSc patients with severe (M(I)II) but none of those with mild impairment were on cyclophosphamide treatment for severe cutaneous fibrosis (P = .02). Conclusion: Severe signs of TMD according to the anamnestic and clinical Helkimo indices were very frequent in SSc patients. J OROFAC PAIN 2010;24:197-202

Lung Diffusing Capacity Relates Better to Short-Term Progression on HRCT Abnormalities Than Spirometry in Mild Asbestosis

Background Pulmonary function tests (PFT), particularly spirometry and lung diffusing capacity for carbon monoxide (DL(CO)), have been considered useful methods for the detection of the progression of interstitial asbestos abnormalities as indicated by high-resolution computed tomography (HRCT). However, it is currently unknown which of these two tests correlates best with anatomical changes over time. Methods In this study, we contrasted longitudinal changes (3-9 years follow-up) in PFTs at rest and during exercise with interstitial abnormalities evaluated by HRCT in 63 ex-workers with mild-to-moderate asbestosis. Results At baseline, patients presented with low-grade asbestosis (Huuskonen classes I-II), and most PFT results were within the limits of normality. In the follow-up, most subjects had normal spirometry, static lung volumes and arterial blood gases. In contrast, frequency of DL(CO) abnormalities almost doubled (P < 0.05). Twenty-three (36.5%) subjects increased the interstitial marks on HRCT. These had significantly larger declines in DL(CO) compared to patients who remained stable (0.88 vs. 0.31 ml/min/mm Hg/year and 3.5 vs. 1.2%/year, respectively; P < 0.05). In contrast, no between-group differences were found for the other functional tests, including spirometry (P > 0.05). Conclusions These data demonstrate that the functional consequences of progression of HRCT abnormalities in mild-to-moderate asbestosis are better reflected by decrements in DL(CO) than by spirometric changes. These results might have important practical implications for medico-legal evaluation of this patient population. Am. J. Ind. Med. 54:185-193, 2011. (c) 2010 Wiley-Liss, Inc.

Comparative Analysis of the Performance of Various Crystalloid Cardioplegic Solutions on Myocardial Protection After Prolonged Cold Ischemia

Introduction. The quality and effectiveness of myocardial protection are fundamental problems to expand the use of and consequently good outcomes of donated hearts for transplantation. Objective. The purpose of this investigation was to compare the cardioprotective effects of Krebs-Henseleit, Bretschneider-HTK, St Thomas, and Celsior solutions using a modified nonrecirculating Langendorff column model of isolated perfused rat heart during prolonged cold storage. Materials and Methods. After removal 36 rat hearts underwent isolated perfusion into a Langendorff apparatus using Krebs-Henseleit solution for a 15-minute period of recovery; we excluded organs that did not maintain an aortic pressure above 100 m Hg. Subsequently, we equally distributed the hearts into four groups according to the cardioprotection solution; group 1, Krebs-Henseleit (control); group II, Bretschneider-HTK; group III, St Thomas; and group IV, Celsior. Each heart received the specific cardioplegic solution at 10 C for 2-hour storage at 20 C, before a 15 minutes perfusion with Krebs-Henseleit solution for recovery and stabilization. After 60 additional minutes of perfusion, every 5 minutes we determined heart rate (HR), coronary flow (CF), left ventricular systolic pressure (LVSP), and positive and negative peak of the first derivative of left ventricular pressure (+dP/dt and dP/dt, respectively). Results. Comparative analysis by Turkey`s test showed the following performances among the groups at 60 minutes of reperfusion: HR: II = IV > III > I; CF: II = IV > I = III; LVSP: IV > I = II = III; +dP/dt: IV > I = II = III; and dP/dt: IV = II > I = II. Conclusion. Cardioprotective solutions generally used in clinical practice are not able to avoid hemodynamic alterations in hearts exposed to prolonged ischemia. Celsior solution showed better performance than Bretschneider-HTK, St Thomas, and Krebs-Henseleit.

Survival in Patients With Idiopathic, Familial, and Anorexigen-Associated Pulmonary Arterial Hypertension in the Modern Management Era

Background-Novel therapies have recently become available for pulmonary arterial hypertension. We conducted a study to characterize mortality in a multicenter prospective cohort of patients diagnosed with idiopathic, familial, or anorexigen-associated pulmonary arterial hypertension in the modern management era. Methods and Results-Between October 2002 and October 2003, 354 consecutive adult patients with idiopathic, familial, or anorexigen-associated pulmonary arterial hypertension (56 incident and 298 prevalent cases) were prospectively enrolled. Patients were followed up for 3 years, and survival rates were analyzed. For incident cases, estimated survival (95% confidence intervals [CIs]) at 1, 2, and 3 years was 85.7% (95% CI, 76.5 to 94.9), 69.6% (95% CI, 57.6 to 81.6), and 54.9% (95% CI, 41.8 to 68.0), respectively. In a combined analysis population (incident patients and prevalent patients diagnosed within 3 years before study entry; n = 190), 1-, 2-, and 3-year survival estimates were 82.9% (95% CI, 72.4 to 95.0), 67.1% (95% CI, 57.1 to 78.8), and 58.2% (95% CI, 49.0 to 69.3), respectively. Individual survival analysis identified the following as significantly and positively associated with survival: female gender, New York Heart Association functional class I/II, greater 6-minute walk distance, lower right atrial pressure, and higher cardiac output. Multivariable analysis showed that being female, having a greater 6-minute walk distance, and exhibiting higher cardiac output were jointly significantly associated with improved survival. Conclusions-In the modern management era, idiopathic, familial, and anorexigen-associated pulmonary arterial hypertension remains a progressive, fatal disease. Mortality is most closely associated with male gender, right ventricular hemodynamic function, and exercise limitation. (Circulation. 2010; 122: 156-163.)

Refractory Angina Cell Therapy (ReACT) Involving Autologous Bone Marrow Cells in Patients Without Left Ventricular Dysfunction: A Possible Role for Monocytes

Autologous bone marrow mononuclear cell (BMMC) transplantation has emerged as a potential therapeutic option for refractory angina patients. Previous studies have shown conflicting myocardium reperfusion results. The present study evaluated safety and efficacy of CellPraxis Refractory Angina Cell Therapy Protocol (ReACT). in which a specific BMMC formulation was administered as the sole therapy for these patients. The phase I/IIa noncontrolled, open label. clinical trial, involved eight patients with refractory angina and viable ischemic myocardium, without left ventricular dysfunction and who were not suitable for conventional myocardial revascularization. ReACT is a surgical procedure involving a single series of multiple injections (40-90 injections, 0.2 ml each) into ischemic areas of the left ventricle. Primary endpoints were Canadian Cardiovascular Society Angina Classification (CCSAC) improvement at 18 months follow-up and myocardium ischemic area reduction (assessed by scintigraphic analysis) at 12 months follow-up, in correlation with a specific BMMC formulation. Almost all patients presented progressive improvement in angina classification beginning 3 months (p = 0.008) postprocedure which was sustained at 18 months follow-up (p = 0.004), as well as objective myocardium ischemic area reduction at 12 months (decrease of 84.4%, p < 0.004). A positive correlation was found between monocyte concentration and CCSAC improvement (r = -0.759, p < 0.05). Improvement in CCSAC, followed by correlated reduction in scintigraphic myocardium ischemic area, strongly suggests neoangiogenesis as the main stem cell action mechanism. The significant correlation between number of monocytes and improvement strongly supports a cell-related effect of ReACT. ReACT appeared safe and effective.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family Hp1(Hs alpha), Hp1(Hs beta) and Hp1(Hs gamma) which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1(Hs alpha), HP1(Hs beta) and HP1(Hs gamma) was determined by real-time RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow. (Int J Biol Markers 2008; 23: 219-24)

Early diagnostic and endoscopic dilatation for the treatment of acquired upper airway stenosis after intubation in children

Background: Upper airway stenosis (UAS) after endotracheal intubation is a common problem in children. Most literature refers to a surgical treatment for these lesions. Laryngotracheal reconstruction and cricotracheal resection are used for low- and high-grade stenosis, but decannulation is not always possible immediately after surgery. Purpose: The aim of this study was to verify the feasibility and results of endoscopic dilatations for treatment of subglottic stenosis. Method: The study encompassed a 12-year retrospective analysis of patients treated for UAS in a tertiary center. All children were symptomatic at the time of the endoscopic diagnosis. The stenosis was graded according to the Myer-Cotton criteria. Endoscopic dilatation was initiated immediately after the diagnosis. Children with grade IV stenosis underwent surgery. Results: Children with tracheal stenosis and no involvement of the subglottic area did not respond to endoscopic dilatations and underwent surgery. There were 45 children with grade I stenosis, 12 with grade II, 7 with grade III, and 4 with grade IV lesions. Patients with grade I, II, and III stenosis were a] I treated by endoscopic dilatations alone and were decannulated when asymptomatic. The average time for decannulation was 18.90 +/- 26.07 months for grade 1, 32.5 +/- 27.08 months for grade 11, and 27.57 +/- 20.60 months for grade III stenosis (P < .01, for grade II vs grade III). Conclusion: Grade I, II, and III subglottic stenoses can be safely managed by early endoscopic dilatations with a high rate of success and low rate of morbidity but require a significantly long period of treatment. (C) 2008 Elsevier Inc. All rights reserved.

Otitis media with effusion and dental occlusion: is there any relationship?

Aim Auditory tube dysfunction is one of the aetiological causes of otitis media. Studies suggest a correlation between otitis media with effusion and dental malocclusion. Our goal was to determine any correlation between dental malocclusion and otitis media with effusion in children with chronic upper airway obstruction due to tonsil and adenoid enlargement. Materials and methods This prospective study evaluated 52 children with otitis media with effusion and 48 without, aged 4.2 to 10.8 years. A questionnaire was answered by the parents about breast or bottle-feeding and bad oral habits. Malocclusion was diagnosed according to Angle`s classification for molar relationships in Classes I, II and III, posterior and anterior cross bite, open and deep bite, and overjet. Results and conclusion The results suggested no correlation between dental malocclusion and otitis media with effusion. Other potential confounders, such as breast or bottle-feeding and oral habits were also not correlated.

Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than CKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with CKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity. (C) 2009 Elsevier B.V. All rights reserved.